RAB18 is a key regulator of GalNAc conjugated siRNA induced silencing in Hep3B cells

Small interfering RNAs (siRNA) therapeutics have developed rapidly in recent years, despite the challenges associated with delivery of large, highly charged nucleic acids. Delivery of siRNA therapeutics to the liver has been established, with conjugation of siRNA to N-acetylgalactosamine (GalNAc) providing durable gene knockdown in hepatocytes following subcutaneous injection. GalNAc binds the asialoglycoprotein receptor (ASGPR) that is highly expressed on hepatocytes and exploits this scavenger receptor to deliver siRNA across the plasma membrane by endocytosis. However, siRNA needs to access the RNA-induced silencing complex (RISC) in the cytoplasm to provide effective gene knockdown and the entire siRNA delivery process is very inefficient, likely due to steps required for endosomal escape, intracellular trafficking, and stability of siRNA. To reveal the cellular factors limiting delivery of siRNA therapeutics, we performed a pooled, genome wide knockout screen based on delivery of GalNAc conjugated siRNA targeting the HPRT1 gene in the human hepatocellular carcinoma line Hep3B. Our primary pooled genome wide knockout screen identified candidate genes that when knocked out significantly enhanced siRNA efficacy in Hep3B cells. Follow-up studies indicate that knockout of one gene in particular, RAB18, improved siRNA efficacy.

Identification and optimization of tunable endosomal escape parameters for enhanced efficacy in peptide-targeted prodrug-loaded nanoparticles

Endosomal escape of nanoparticles (NPs) is a weighty consideration for engineering successful nanomedicines.

Cell-Penetrating Peptides: Emerging Tools for mRNA Delivery

Messenger RNAs (mRNAs) were previously shown to have great potential for preventive vaccination against infectious diseases and therapeutic applications in the treatment of cancers and genetic diseases. Delivery systems for mRNAs, including lipid- and polymer-based carriers, are being developed for improving mRNA bioavailability. Among these systems, cell-penetrating peptides (CPPs) of 4–40 amino acids have emerged as powerful tools for mRNA delivery, which were originally developed to deliver membrane-impermeable drugs, peptides, proteins, and nucleic acids to cells and tissues. Various functionalities can be integrated into CPPs by tuning the composition and sequence of natural and non-natural amino acids for mRNA delivery. With the employment of CPPs, improved endosomal escape efficiencies, selective targeting of dendritic cells (DCs), modulation of endosomal pathways for efficient antigen presentation by DCs, and effective mRNA delivery to the lungs by dry powder inhalation have been reported; additionally, they have been found to prolong protein expression by intracellular stabilization of mRNA. This review highlights the distinctive features of CPP-based mRNA delivery systems.

Histidine-Tagged Folate-Targeted Gold Nanoparticles for Enhanced Transgene Expression in Breast Cancer Cells In Vitro

Nanotechnology has emerged as a promising treatment strategy in gene therapy, especially against diseases such as cancer. Gold nanoparticles (AuNPs) are regarded as favorable gene delivery vehicles due to their low toxicity, ease of synthesis and ability to be functionalized. This study aimed to prepare functionalized AuNPs (FAuNPs) and evaluate their folate-targeted and nontargeted pCMV-Luc-DNA delivery in breast cancer cells in vitro. CS was added to induce stability and positive charges to the AuNPs (Au-CS), histidine (Au-CS-His) to enhance endosomal escape and folic acid for folate-receptor targeting (Au-CS-FA-His). The FAuNP:pDNA nanocomplexes possessed favorable sizes (<135 nm) and zeta potentials (<−20 mV), strong compaction efficiency and were capable of pDNA protection against nuclease degradation. These nanocomplexes showed minimal cytotoxicity (>73% cell viability) and enhanced transgene activity. The influence of His was notable in the HER2 overexpressing SKBR3 cells, which produced higher gene expression. Furthermore, the FA-targeted nanocomplexes enhanced receptor-mediated endocytosis, especially in MCF-7 cells, as confirmed by the receptor competition assay. While the role of His may need further optimization, the results achieved suggest that these FAuNPs may be suitable gene delivery vehicles for breast cancer therapeutics.

RNA Drug Delivery Using Biogenic Nanovehicles for Cancer Therapy

RNA-based therapies have been promising method for treating all kinds of diseases, and four siRNA-based drugs and two mRNA-based drugs have been approved and are on the market now. However, none of them is applied for cancer treatment. This is not only because of the complexity of the tumor microenvironment, but also due to the intrinsic obstacles of RNAs. Until now, all kinds of strategies have been developed to improve the performance of RNAs for cancer therapy, especially the nanoparticle-based ones using biogenic materials. They are much more compatible with less toxicity compared to the ones using synthetic polymers, and the most widely studied biogenic materials are oligonucleotides, exosomes, and cell membranes. Particular characteristics make them show different capacities in internalization and endosomal escape as well as specific targeting. In this paper, we systematically summarize the RNA-based nano-delivery systems using biogenic materials for cancer therapy, and we believe this review will provide a valuable reference for researchers involved in the field of biogenic delivery and RNA-based therapies for cancer treatment.

Quantitative intracellular retention of delivered RNAs through optimized cell fixation and immuno-staining.

Detection of nucleic acids within sub-cellular compartments is key to understanding their function. Determining the intracellular distribution of nucleic acids requires quantitative retention and estimation of their association with different organelles by immunofluorescence microscopy. This is particularly important for the delivery of nucleic acid therapeutics which depends on endocytic uptake and endosomal escape. However, the current protocols fail to preserve the majority of exogenously delivered nucleic acids in the cytoplasm. To solve this problem, by monitoring Cy5-labeled mRNA delivered to primary human adipocytes via lipid nanoparticles (LNP), we optimized cell fixation, permeabilization and immuno-staining of a number of organelle markers, achieving quantitative retention of mRNA and allowing visualization of levels which escape detection using conventional procedures. The optimized protocol proved effective on exogenously delivered siRNA, miRNA, as well as endogenous miRNA. Our protocol is compatible with RNA probes of single molecule fluorescence in-situ hybridization (smFISH) and molecular beacon, thus demonstrating that it is broadly applicable to study a variety of nucleic acids in cultured cells.

Non-Cationic RGD-Containing Protein Nanocarrier for Tumor-Targeted siRNA Delivery

Despite the recent successes in siRNA therapeutics, targeted delivery beyond the liver remains the major hurdle for the widespread application of siRNA in vivo. Current cationic liposome or polymer-based delivery agents are restricted to the liver and suffer from off-target effects, poor clearance, low serum stability, and high toxicity. In this study, we genetically engineered a non-cationic non-viral tumor-targeted universal siRNA nanocarrier (MW 26 KDa). This protein nanocarrier consists of three function domains: a dsRNA binding domain (dsRBD) (from human protein kinase R) for any siRNA binding, 18-histidine for endosome escape, and two RGD peptides at the N- and C-termini for targeting tumor and tumor neovasculature. We showed that cloned dual-RGD-dsRBD-18his (dual-RGD) protein protects siRNA against RNases, induces effective siRNA endosomal escape, specifically targets integrin αvβ3 expressing cells in vitro, and homes siRNA to tumors in vivo. The delivered siRNA leads to target gene knockdown in the cell lines and tumor xenografts with low toxicity. This multifunctional and biomimetic siRNA carrier is biodegradable, has low toxicity, is suitable for mass production by fermentation, and is serum stable, holding great potential to provide a widely applicable siRNA carrier for tumor-targeted siRNA delivery.

Endosomal escape of delivered mRNA from endosomal recycling tubules visualized at the nanoscale

Delivery of exogenous mRNA using lipid nanoparticles (LNPs) is a promising strategy for therapeutics. However, a bottleneck remains in the poor understanding of the parameters that correlate with endosomal escape versus cytotoxicity. To address this problem, we compared the endosomal distribution of six LNP-mRNA formulations of diverse chemical composition and efficacy, similar to those used in mRNA-based vaccines, in primary human adipocytes, fibroblasts, and HeLa cells. Surprisingly, we found that total uptake is not a sufficient predictor of delivery, and different LNPs vary considerably in endosomal distributions. Prolonged uptake impaired endosomal acidification, a sign of cytotoxicity, and caused mRNA to accumulate in compartments defective in cargo transport and unproductive for delivery. In contrast, early endocytic/recycling compartments have the highest probability for mRNA escape. By using super-resolution microscopy, we could resolve a single LNP-mRNA within subendosomal compartments and capture events of mRNA escape from endosomal recycling tubules. Our results change the view of the mechanisms of endosomal escape and define quantitative parameters to guide the development of mRNA formulations toward higher efficacy and lower cytotoxicity.